Immunomodulator, immunomodulator food

ABSTRACT

An immunomodulator having suppressive activity on IgE antibody production is provided, comprising bacterial cells, or their decomposition materials. It can be taken as food. Bacterial cells such as Corynebacterium, Brevibacterium, Microbacterium, or bacterial cells of mutant strains of these bacteria, or decomposition products of these bacteria are used.

FIELD OF THE INVENTION

[0001] The present invention relates to a decomposition product ofbacterial cell walls, an immunomodulator, consisting of bacterial cellsor their decomposition materials which can suppress selectively IgEantibody production, and to provide their production methods and foodscontaining them.

[0002] 1. Related Art

[0003] Immediate type allergy or atopy, such as pollen allergy, asthma,atopic dermatitis, food allergy, are known to be mediated by antibody ofIgE isotype immunoglobulin. Symptoms of atopy are elicited when the IgEantibodies react to antigen, resulting in release of variousphysiologically active substances such as histamine, serotonin,leucotrienes and others.

[0004] For the prevention and treatment of immediate type allergy, drugsgenerally used today are those which are antagonistic to the mediators,for instance, antihistaminics, drugs which inhibit release of mediatorsfrom mast cells, and corticosteroids which inhibit activation of variousimmunocytes resulting in inhibition of cytokine production.

[0005] Since antibody which mediates immediate allergy is IgE antibody,an agent which suppress IgE antibody production can be thought to beeffective for the treatment of immediate type allergy. However, thereare no effective drugs which suppress only IgE isotype antibody withoutsuppressing other isotypes such as IgG, IgA so on, which are importantfor body defense mechanisms to various infectious diseases.

[0006] 2. Summary of the invention

[0007] Accordingly, an object of the present invention is to provide animmunomodulator having suppressive activity on IgE production withoutany suppressive effect on other isotypes of antibody.

[0008] As a result of extensive investigation in order to solve theabove-mentioned problem, the present investigators have unexpectedlyfound firstly that bacterial cells of Corynebacterium, Brevibacterium,and Microbacterium species, and mutant strains derived from the speciesare able to suppress selectively IgE type antibody production by oraladministration of these bacterial cells without any suppressive effecton the production of other isotypes even if enhancing these antibodyproduction, and secondly that a decomposition product of above mentionedbacteria by enzymatic treatment and such, has more potentimmunomodulative activity than that of the bacterial cells bythemselves. These findings have led to the completion of the presentinvention.

[0009] That is, the present invention is to provide an immunomodulator,an immunomodulator food, immunomodulator drink containing bacterialcells belonging to Corynebacterium, Brevibacterium, and Microbacteriumspecies, and mutant strains derived from the species, and adecomposition material of above mentioned bacteria by enzymatictreatment and such.

[0010] The invention provides a method of modulating the immunologicalfunction by administering orally a pharmacologically effective amount ofbacterial cells or their decomposition materials to human being.

[0011] The invention provides a bacterial cell wall decompositionproduct (or decomposition material) having an immunomodulative activity,which is produced by dissolving the cell wall of at least one selectedfrom the group consisting of bacteria belonging to Corynebacteruimgenera, Brevibacterium genera and Microbacterium genera and mutantstrains of these bacteria.

[0012] The invention provides a process for manufacturing theabove-shown bacterial cell wall decomposition product, which comprisesdissolving the cell wall of at least one selected from the groupconsisting of bacteria belonging to Corynebacteruim genera,Brevibacterium genera and Microbacterium genera and mutant strains ofthese bacteria.

[0013] It is preferably to the process to dissolve the cell walls withone of gulcosidase and endopeptidase in the medium of an isotonicsolution, separate the protoplast generated by the enzymatic digestionof the bacterial cells from the medium, and furthermore enzymaticallytreat the dissolved material of the bacterial cells with the other ofendopeptidase and glucosidase.

[0014] It is preferably to dissolve the cell walls with one ofgulcosidase and endopeptidase in the medium of an isotonic solution(biological saline), centrifuge the solution to obtain the supernatantand enzymatically treat the supernatant with the other of endopeptidaseand glucosidase.

[0015] The decomposition production of the invention may be obtainedwhen at least 30% or 29% of the bacterial cells have been decomposed.This decomposition extent can be observed in terms of determination ofdissolved hexosamine or glucosamine, which is present in thesupernatant. Alternatively, it can be observed in terms of opticaldensity of the bacterial suspension. The invention may reach at least25% of optical density.

[0016] The invention provides an immunomodulator food or drinkcontaining the bacterial cell wall decomposition product of theinvention.

[0017] The invention provides a method of treating or preventingallergy, which comprises administering a pharmacologically effectiveamount of the decomposition product of the invention to a personsuffering from allergy, and use of the decomposition product formanufacturing immunomodulator.

[0018] The invention relates to use of bacteria belonging toCorynebacteruim genera, Brevibacterium genera and Microbacterium generaand mutant strains of these bacteria. That is, the invention provides amethod of treating or preventing allergy, which comprises administeringa pharmacologically effective amount of the bacteria to a personsuffering from allergy; use of the bacteria for manufacturingimmunomodulator; an Immunomodulator food or drink containing thebacteria; and a method of effecting immunomodulation, which comprisesadministering a pharmacologically effective amount of the bacteria to aperson suffering from immunodeficiency.

[0019] It is preferred to the production of the decomposition productthat the bacterial cell wall is digested with glucosidase and/orendopeptidase; a bacterium of Corynebacteruim genus is Corynebacteruimglutamicum or Corynebacteruim herculis; a bacterium of Brevibacteriumgenus is Brevibacterium flavum; a bacterium of Microbacterium genus isMicrobacterium ammoniaphilum; the glycosidase is egg white lysozyme orN-acetylmuramidase SG; the decomposition is performed by induction oflysogenic bacteriophage; the decomposition is performed by autolysis;the endopeptidase is bromelain; or the endopeptidase is one selectedfrom the group consisting of seratiopeptidase, protease produced byStreptomyces griseus (K-1 strain), protease produced by Bacillussubtilis, Seaproze S produced by Armillaria mellea (Naratake inJapanese), trypsin, Achromobacter protease I and Grifola frondosa(Maitake in Japanese) metaloendopeptidase.

[0020] It is preferable that the immunomodulative bacterial cells areproducts produced by culturing cells of strains belonging toCorynebacterium, Brevibacterium, and Microbacterium species, and mutantstrains derived from the species, heating the culture to kill the cells,harvesting the killed cells and dried. As for products ofimmunomodulative decomposition materials made of above mentionedbacteria, cultured cells are decomposed by autolysis, induction oflysogenic bacteriophage, and enzymatic treatment using cell walldigesting enzymes.

[0021] In the invention, decomposition material is defined by ageneration of soluble hexosamine at least 30% of total bacterialhexosamine.

DETAILED DESCRIPTION OF THE INVENTION

[0022] An immuno-adjuvant is called for the substances which enhanceantibody formation by injecting them mixed with antigen to animals.Killed bacterial cells of Micobacterim tuberculosis have been knownhaving immuno-adjuvant activity. Results of study for clarifying aneffective component of the adjuvant activity revealed that the smallestcomponent was muramyl-L-alanyl-D-isogultamine (MDP), a component ofpeptidoglycan of bacterial cell wall. The existence of MDP is also knownin all the bacterial cells other than bacteria of Micobacterimtuberculosis, including pathogenic and also non-pathogenic bacteria,irrespective to classification by gram-positive and gram-negativestaining. In fact, bacterial cell wall fractions containing MDP showedadjuvant activity in almost all bacteria studied (Kotani, S. Seikagaku48,1081-1107 1976 in Japanese).

[0023] A mucopeptide layer of bacterial cell wall is consisted of longglucoside chains polymerized at β-1,4 linkage of N-acetylglucosamine andmuramic acid, and of peptide linkage which links calboxylic residue ofmuramic acid with alanine or L-glycine,- D-glutamic acid, L-lysine ormesodiaminopimeric acid, D-alanine in this turn. The last D-alaninemakes peptide bond with a carboxic residue of another D-glutaminc acid,lysine and mesodiaminopimeric acid which is in the peptide chainoriginated from a neighboring glucoside chain. Number of amino acids inthe peptide which links two neighboring glucoside chain are, somewhatdifferent dependent on species of bacteria, 6 to 7 amino-acids.Supposing that many glucoside chains arranged in parallel with eachother are the warps, the peptide chains which bind the glucoside chainseach other are the woofs, therefore, both the woofs and the warps make anet-like structure and named peptidoglycan. The peptidoglycan surroundscytoplasm of the bacterial cell and makes strong wall which protect thebacterial cell from damage by physical change of osmotic pressure.

[0024] The enzymes which solubilize the bacterial cell walls belong tothe hydrolyze enzyme. Depending on their actin mechanisms, they aredivided to three categories. One is glycosidase which hydrolyzecarbohydrate linkage (for an instance egg-white lysozyme), others areendopeptidase which hydrolyze peptide bonds of peptidoglycan and amidasewhich hydrolyze a bond between muramic asid and amino acid.

[0025] Accordingly, MDP containing components are solubilized frombacterial cell wall by above-mentioned hydrolyze enzymes excludingamidase.

[0026] Being based on these background, various researches have beenperformed for the purpose of potentiation of immune status of the host.As the results, it has been reported studies on the resistance to tumor(Bogdanov IG. et al. Antitumor glycopeptide from Lactobacillusbulgaricus cell wall FEBS LETT:57(3) 259-2611975), activation ofmacrophage function and cellular immunity concerning anti-infectiousimmunity, and enhanced production of IgG antibody that plays importantrole for the resistance to bacterial and viral infections (Nammba Y etal. Effect of oral administration of lysozyme or digested cell wall onimmunostimulation in guinea pigs. Infect Immun 31:580-583 1981). As forrelationships between chemical structure and biological activity,bacterial cell wall and its enzymatic digested material have beenstudied (Kotani, S. Seikagaku 48,1081-1107 1976 in Japanese). However,no findings have been reported today as for production of IgE antibodywhich induces allergic disease such as atopy.

[0027] In the present invention, the bacteria are not necessarilyspecial bacteria, However, in view of the safety and the utilization ofwaste material, amino acid producing bacteria, for example,Corynebacterium glutamicum, Corynebacterium herculis, Corynebacteriumfermentum, Brevibacterium fluvum and Microbcacterium ammoniaphilum canbe mentioned.

[0028] The present inventors investigated effect of above-mentionedbacterial cells, a mutant strain of these bacteria, and enzymaticallydigested materials of these bacterial cells on IgE antibody productionby oral administration, and have found that each bacterium suppressedIgE antibody production by administering heat-killed bacteria,enzymatically digested materials of the bacteria containing MDP, andthat the IgE production suppressing activity of bacterial cells waspotentiated by digesting the cells with cell-wall digesting enzymes.

[0029] Culture of bacteria belonging to genera of Corynebacterium,Brevibacterium and Microbacterium can be made by a known method in anappropriate medium. Any form of bacterial cell preparations can be usedas a starting material such as culture itself, cenrtifuged cells,freeze-dried cells, heat-killed cells and spray-dried cells for thepurpose of production of the final raw products. Heat-killed cells orspray dried cells can be used as the IgE production immunomodulativematerial having suppresive effect on IgE production.

[0030] Production of immunomodulative decomposition material startingfrom bacterial cell preparations is performed by solubilizing bacterialcell wall by means of using cell wall digesting enzymes, induction oflysogenic bacteriophage and autolysis using endogenous autolyticenzymes.

[0031] Of the methods mentioned above, a efficient method ofdecomposition of bacterial cell wall is the method using glycosidase and/or protease of endopeptidase type. The present invention revealed thatthe decomposition material produced by use of both of glycosidase andproteinase had more potent immunomodulative activity than that producedby use of either one of the two enzymes. Comparing the activity producedby two kinds of the enzymes, glycosidase is preferential. As an exampleof glycosidase, egg-white lysozyme, bacterial lysozyme andN-acetylmulamidase SG are mentioned. Of them, egg-white lysozyme isrecommended from a stand point of safety of the product because thisenzyme is used as a food additive.

[0032] As an example of the endopeptidase, bromelain, seratiopeptidase,protease produced by Streptomyces griseus K-1 (Pronase®), proteaseproduced by Bacillus subtilis (Nagase®), Seaprose S produced byArmillaria mellea (Naratake in Japanese), Acromobacter protease 1(lysyl-endopeptidase) and metalloendopeptidase produced by Grifolafrondosa (Maitake in Japanese) can be mentioned. Of these, bromelain isrecommended from a stand point of safety of the product because thisenzyme is used as a food additive.

[0033] Sensitivity of bacterial cells to glycosidase digestion differsdepend on a bacterial strain. The sensitivity can be enhanced byaddition of penicillin or glycine in culture medium as described inExamples 1 and 2. Enzyme treatment is conducted under nearly optimal pHof the respective enzymes. The treatment by glycosidase andendopeptidase is performed under nearly neutral pH, i.e., pH 5 to 8. Anamount of enzyme added to 1 gm of dry bacterial cells is 0.01 to 10 mg,Temperature of enzyme treatment can be conducted at room temperature to70° C. DNase and RNase at concentrations of 10 to 500 μg/ml can be addedto the reaction mixture to reduce viscosity of the mixture.

[0034] Decomposition of bacterial cell by an induction of lysogenicbacteriophage can be done by UV irradiation of each of bacterial cultureof Corynebacterium, Brevibacterium and Microbacterium. Decomposition ofbacterial cell by autolysis can be performed by incubation of bacterialcell suspended in purified water at about 50° C. for 1 to 3 days.

[0035] A definite method for production of the immunomodulativedecomposed material of the present invention is, for example, thefollowing. Corynebacterium glutamicum is cultured in appropriate medium.The culture is centrifuged to collect bacterial cells. The cells aresuspended in physiological isotonic solution such as physiologicalsaline, and then either of glycosidase such as egg-white lysozyme orendopeptidase such as bromelain is added to the suspension to solubilizethe bacterial cell wall. Bacterial cytoplasmic membrane and cytoplasmsurrounded by the cytoplasmic membrane are existed as plotoplast by thisstage of enzyme treatment. The plotoplast is sedimented bycentrifugation and discarded. Then, the supernatant, which contains MDPcontaining cell wall components, is treated with another enzyme, i.e.,endopeptidase (if enzyme used at the first step is glycosidase) such asbromeline or glycosidase (if enzyme used at the first step isendopeptidase) such as egg white lysozyme.

[0036] A preferable method is the following. At first, bacterial cellwall is solubilized by treatment with glycosidase and plotoplast issedimented and discarded and then endopeptidase treatment of thesupernatant solution is conducted. Elimination of plotoplast effectivelypurify the active decomposed material of present invention as inExample. The immunomodulative active decomposition material can beprecipitated by adding ethanol or acetone to the supernatant.

[0037] Other method of preparation of the present invention is thefollowing. Corynebacterium glutamicum is cultured and the bacterialcells are harvested and suspended in purified water. Then at firstglycosidase is added to the suspension and incubated to lyse cell walland then endopeptidase is added to the suspension and incubated.

[0038] The process of decomposition of bacterial cells is measured bythe estimation of optical density of bacterial suspention at wave lengthof 600 to 660 nm as described in Example 7 or by hexosaminedetermination of supernatant solution after centrifugation of bacterialsuspention as described in Example 4. Hexosamine determination can beconducted by ordinally methods such as Elson-Morgan Method and Reissig,Strominger and Leloir Method.

[0039] An enhanced activity on immunomodulation owing to decompositionof bacterial cells is observed when at least 30% of bacterial cells havebeen decomposed in terms of dissolved hexosamine determination.Alternatively, at least 25% reduction of optical density of thebacterial suspension is preferably found for this purpose as in Example4.

[0040] A standard oral daily dose of the immunomodulative bacterialcells and their decomposition material of the present invention isbetween 0.2 and 2 g. The desirable dose is about 1 g equivalent tokilled dry cells.

[0041] When the immunomodulative decomposition material of the presentinvention is applied as a main agent, it can orally be used in powder,tablets, dispersion, capsules, confectionery, bread, noodles, drinks orthe like. These products can be made by ordinary production methods.

EXAMPLES

[0042] The present invention is illustrated more specifically by givingthe following examples. By the way, in Examples, % and parts are both onthe weight basis.

Example 1

[0043] (Decomposition Material By Enzyme Treatment)

[0044]Corynebacterium glutamicum IAM 12435 was inoculated into anL-shaped tube containing 10 ml of nutrient broth, and incubated whilebeing shaken at 30° C. for 7 hours. One ml of this culture wasinoculated in each of 10 conical flask (Erlenmeyer flask) having acapacity of 500 ml and containing 100 ml of a medium of Miura et al.(Hakko Kogaku Zasshi, Vol. 41, No. 5, pp. 275-281, 1963), and incubatedwhile being shaken at 30° C. for 24 hours. Penicillin G was added at aconcentration of 0.2 U/ml in the logarithmic phase of the culture. Thecells were harvested by centrifugation to obtain approximately 5 g ofwet cells. The wet cells were washed by centrifugation once withphysiological saline. Then, 1 liter of physiological saline was added tothe sediment to suspend the cells. Five mg of egg-white lysozyme wasadded thereto while being stirred. After the stirring at 37° C. for onehour, 5 mg of seratiopeptidase was added and incubated at 37° C. for onehour under stirring. Thus, the decomposed material was prepared. Thiswas heated at 80° C. for 30 min and freeze-dried.5 g of dry material wasobtained.

Example 2

[0045] (Decomposition Material By Enzymatic Digestion and Purification)

[0046]Corynebacterium glutamicum IAM 12435 was inoculated into anL-shaped tube containing 10 ml of nutrient broth, and incubated whilebeing shaken at 30° C. for 7 hours. One ml of this culture wasinoculated in a conical flask (Erlenmeyer flask) having a capacity of500 ml and containing 100 ml of nutrient broth and incubated while beingshaken at 30° C. for 8 hours. The whole culture in the flask wasinoculated into a jar fermenter of inner volume of 2 liter andcontaining 1.5 liter of Mimura's medium and incubated at 30° C. for 20hours under airation. Penicillin G was added at a concentration of 0.2U/ml in the logarithmic phase of the culture. The cells were harvestedby centrifugation to obtain 7.5 g of wet cells. After washing the wetcells with physiological saline by centrifugation, the cells wereresuspended with 200 ml of physiological saline and added with 5 mg ofegg-white lysozyme while being stirred for one hour at 37° C. Assessmentof lysozyme treatment was performed by an estimation of optical density(OD) at 660 nm for the samples obtained before and after the treatment.OD values of 40 fold diluted suspensions before and after egg-whitelysozyme treatment were 1.20 and 0.77, respectively. The reduction ofoptical density is calculated as 35.8%. The incubation mixture wascentrifuged at 13000 rpm for 30 min. The sediment containing plotoplastswas discarded and the supernatant was obtained. The supernatant wasadded with 5 mg of seratiopeptidase and incubated at 37° C. for one hourunder stirring. Thus, the decomposition material was prepared. This washeated at 80° C. for 30 min, desalted by dialysis and freeze-dried toobtain 0.2 g of dry material.

Example 3

[0047] (Decomposition Material By Enzymatic Digestion and Purification)

[0048] The immunomodulative decomposition material produced according tothe method of example 2 in which 5 mg of bromelaine was used instead ofthe same amount of seratiopeptidase. Assessment of lysozyme treatmentwas performed by an estimation of OD at 660 nm for the samples obtainedbefore and after the treatment. OD values of 40 fold diluted suspensionsbefore and after egg-white lysozyme treatment were 1.05 and 0.76,respectively. The reduction of optical density is calculated as 27.6%.Thus, 0.3 g of the dry material was obtained.

Example 4

[0049] (Decomposition Material By Enzymatic Digestion)

[0050] One kg of freeze-dried bacterial cells of Corynebacteriumglutamicum IAM 12435 was suspended with 12 l of physiological saline.Then, 100 ml physiological saline containing 5 g of egg-white lysozymewas added to the suspension. After stirring for 1 hour at 30° C., 200 mlof physiological saline containing 5 g of bromelain was added andincubated at 30° C. for 1 hour under stirring to make the decomposedmaterial of the present invention. The material was spray-dried toobtain 1 kg of the dry material.

[0051] Assessment of decomposition was performed by determiningsolubilized hexosamine from bacterial cells. Two-tenth ml of bacterialsuspension was diluted five fold with purified water and centrifuged at16000 rpm for 10 min and a supernatant solution was separated.Hexosamine contents of the five fold diluted suspension and those in thesupernatant solution, before and after lysozyme and bromelain treatmentwere analyzed by the method of Reissig, Strominger and Leloir method.The results of estimation of each of the bacterial suspension,supernatants before lysozyme treatment and after bromelain treatmentwere 1820, 0, and 535 μl/ml, respectively. The amount of decomposedbacteria is calculated as 29.4% from 535/1820.

Example 5

[0052] (Decomposition Material By Enzymatic Digestion)

[0053] The same process as in Example 4 was repeated, except thatfreeze-dried bacterial cells of Corynebacterium glutamicum ATCC 13032was used instead of that of Corynebacterium glutamicum IAM 12435, toobtain 1 kg of the above-mentioned dry decomposed material.

Example 6

[0054] (Decomposition Material By Autolysis)

[0055] One kg of freeze-dried bacterial cells of CorynebacteriumGlutamicum ATCC 13032 was suspended in 20 l of purified water and thesuspension was kept at 50° C. for 24 hours to autolysis the bacterialcells. The autolysate was spray-dried and 1 kg of the dry decomposedmaterial was obtained.

Example 7

[0056] (Decomposition Material By Cell Lysis According to LysogenicBacteriophage Induction)

[0057] Bacterial cell lysis by inducing lysogenic bacteriophage ofCorynebacterium was performed as the following.

[0058]Corynebacterium glutamicum ATCC 31830 was inoculated into conicalflask (Erlenmeyer flask) having a capacity of 500 ml and containing 100ml of nutrient broth and incubated while being shaken at 30° C. for 24hours. After harvesting the bacterial cells through centrifugation, thecells were suspended in phosphate buffered physiological saline. Twentymilliliters of the bacterial cell suspension were poured into a Petridish of 9 cm diameter and exposed to UV light for 20 sec under stirringusing 15 W germicidal UV lamp at a distance of 40 cm. After theirradiation, the suspension was added with 20 ml of nutrient broth of 2fold concentration of an ordinary broth and cultured for 2 hours at 30°C. under shaking. The optical density at 660 nm of the bacterialsuspension at the beginning of cultivation was 5.0, and that after 2hour incubation was 2.2, demonstrating bacterial lysis.

Example 8

[0059] (Decomposition Material By Enzymatic Digestion)

[0060]Corynebacterium glutamicum ATCC 15354 was inoculated in each of 20conical flasks containing 100 ml of a nutrient broth, and incubatedwhile being shaken at 30° C. for 18 hours. The cells were harvested bycentrifugation to obtain approximately 3 g of wet cells. The wet cellswere washed by centrifugation once with physiological saline. Then, 500ml of physiological saline was added to the sediment to suspend thecells. Four mg of egg-white lysozyme was added thereto while beingstirred. After the stirring at 37° C. for 2 hours, 4 mg ofseratiopeptidase was added and further incubated at 37° C. for 2 hoursunder stirring to obtain the decomposition material of the presentinvention. This was heated at 80° C. for 30 min and freeze-dried toobtain 3 g of the dry material.

Example 9

[0061] (Suppressive Activities on IgE Production of Heat-killed Cells,Heat-killed and Enzymatically Decomposed Cells and Heat-killed,Enzymatically Decomposed and Then Purified Fraction)

[0062] One group consisted of 6 female BALB/c mice of 6 weeks old werefed with feed which contained each of heat-killed bacterial cells ofCorynebacterium glutamicum, preparations of the present inventionmentioned in Examples 3 and 4. After two week feeding, mice wereimmunized with 0.1 ml of physiological saline solution containing 10 μgof egg albumin and 1 mg of alum. Then, blood of each mice was taken byretro-orbital puncture 14 days after the immunization and serum wasseparated. Six sera of each group were pooled and IgE antibody titer ofthe pooled sera was measured by rat PCA reaction. Briefly, 15 to 20 weekold male SD rats were shaved on the back and 0.1 ml of serum previouslydiluted with physiological saline was intradermally injected into theback of 2 recipient rats under pentobarbital anesthesia. After 24 hoursof intradermal injection of sera, one ml of physiological salinesolution containing 1 mg of egg-albumin and 10 mg of Evans blue dye wasintravenously injected to the rats under pentobarbital anesthesia.Thirty minutes after the intravenous injection, diameters of blue spotsappeared on the back of the site of serum injection were measured. Themean diameter more than 5 mm was judged as a positive reaction. Anantibody titer was expressed as a reciprocal of serum dilution. Of thetwo antibody titers obtained from two recipient rats, the higher one wasadopted as the antibody titer for each of the pooled sera. The resultswere shown in Table 1. In the table, Judgement of immunomodulativeactivity was significant in case there was a difference more than 2serial dilution between control group and fed group and + was added forthe difference of each one serial dilution. TABLE 1 Animal PCA antibodyImmunomodulator Dose (%) group No. titer Judgement Control 0 1 80 − Heatkilled cell 0.01 2 80 − 0.1 3 40 − 1 4 10 ++ Enzymatically 0.01 5 40 −decomposed whole 0.1 6 10 ++ cell (Example 4) 1 7  0 ++++ Enzymatically0.001 8 20 + decomposed 0.01 9 10 ++ active fraction 0.1 10   0 ++++(Example 3) 1 11   0 ++++

[0063] As shown in Table 1, an administration to mice with heat killedcells or its decomposed materials prior to an antigenic stimulationsuppressed IgE antibody production. The results have revealed that theadministration have influenced on the immunological reactivity of bodyor the constitution. The result that the stronger effect was observed byan administration of the decomposed material of Example 4 in comparisonwith killed cell alone showed that enzymatic treatment of bacterialcells fortified the suppressive activity, furthermore, that thesuppressive activity for IgE antibody production was the most potent ingroups administered with the decomposition material of Example 3revealed that the active decomposition material was mainly constituentsof bacterial cell wall.

Example 10

[0064] (Enhancing Activities on IgG Production of Heat-killed Cells,Heat-killed and Enzymatically Decomposed Cells and Heat-killed,Enzymatically Decomposed and Then Purified Fraction)

[0065] Effect of immunomodulative decomposition material on IgG antibodyproduction was examined by ELISA of anti-egg-albumin IgG antibodyestimation in the sera of mice of Example 9. Wells of micro-plate(96-well flat bottom ELISA plate, Coster, Cambridge, Mass., USA) wasadded with 50 μl of egg-albumin solution (0.1 mg/ml in 0.05 M carbonatebuffer, pH 9.5), and kept at 4C overnight. After washing the wells witha solution containing 0.9% NaCl and 0.05% Tween 20, 200 μl of bovinealbumin solution (1 mg/ml BSA in phosphate buffered physiological saline(PBS Nissui Pharmaceutical Co.) was added to the wells and incubated at37° C. for one hour to block any nonspecific binding. Next, afterwashing, 50 μl of sera of mice prepared in Example 9, previously dilutedwith PBS solution containing 10 mg/ml BSA , 0.05% Tween 20 and 3% NaCl(Solution A) was added to the wells and kept at 37° C. for 1 hour. Then,after washing the wells, the wells were added with 50 μl of peroxidaselabeled anti-mouse IgG goat sera previously diluted 10000 fold withsolution A and incubated at 37° C. for 1 hour, and washed. Then, each ofwells was added with 100 μl of substrate solution (O-phenylenediamine 40mg, 20 μl of 30% hydrogen peroxide in 100 ml of citrate-sodium phosphatebuffer) and kept at room temperature. Optical density at 492 nm of thewells was measured.

[0066] Amount of antibody was measured by a calibration curve made byuse of anti-ovalbumin mouse IgG antibody which was purified by ovalbuminbounded Sepharose 4B column affinity chromatography of antisera obtainedfrom mice immunized with ovalbumin-and Freund's complete adjuvant.

[0067] The results of antibody measurement in the serum samples used inExample 9 were shown in Table 2. TABLE 2 Animal Anti ovalbuminimmunomodulator Dose (%) group No. IgG (ng/ml) Control 0 1 300 Heatkilled cell 0.01 2 310 0.1 3 310 1 4 640 Enzymatically 0.01 5 285decomposed whole 0.1 6 490 Cell (Example 4) 1 7 1015  Enzymatically0.001 8 300 decomposed active 0.01 9 520 fraction 0.1 10  1820  (Example3) 1 11  1910 

[0068] The result revealed that administration of bacterial cells ofCorynebacteriaum glutamicum and also its enzymatically decomposedmaterials enhanced production of IgG antibody.

Example 11

[0069] (Safety Studies on the Immunomodulator)

[0070] Safety of immunomodulative decomposed material having suppressiveactivity on IgE antibody production As for single dose oral toxicitystudy, a group of 5 male rats of 4 weeks old SD strain was orallyadministered with enzymatically decomposed material prepared by themethod described in Example 4 at a dose of 2 g/kg body weight. As forrepeated oral toxicity, a group of 10 male rats of 4 weeks old SD strainwas fed with food containing at a concentration of 5% for 4 weeks. Theboth groups of rats were compared with rats of respective control group.There were no toxic sign after single oral dose of the test sample, andas shown in Table 3, there were no changes in increase in body weightand behavior between the test group and control. TABLE 3 Body weight (g)Body weight (g) at beginning after 4 week Feed of feeding feeding 5%(w/w) enzymatically 220.5 ± 10.6 401.4 ± 18.8 decomposed material (inExample 4) containing feed Control 222.8 ± 10.9 403.3 ± 21.8

Example 12

[0071] (Tablet Type Food Containing the Immunomodulator)

[0072] Ninety milligrams of immunomodulative decomposition material ofCorynebacterum glutamicum bacterial cells using egg-white lysozyme andbromelain obtained in Example 4, 30 mg of starch, 180 mg of Avicell(Cellulose) were mixed and a tablet-type food was produced in a usualmanner such that one tablet was 300 mg.

Example 13

[0073] (Drink Containing the Immunomodulator)

[0074] Purified water was added to two parts of decomposition materialof Corynebacterum glutamicum bacterial cells using egg-white lysozymeand bromelain obtained in Example 4, 10 parts of cocoa butter, 7 partsof granulated sugar, 7 parts of milk and 0.05 part of emulsifying agentto adjust the total amount to 100 parts, and a cocoa drink was producedin a usual manner.

Example 14

[0075] (Tablet Type Food Containing the Immunomodulator)

[0076] Ninety milligrams of enzymatically decomposed material ofCorynebacterum ammoniaphilum bacterial cells using egg-white lysozymeand bromelain obtained in Example 8, 30 mg of starch, 180 mg of Avicell(Cellulose, Asahi Chemical Industry Co., Ltd.) were mixed and atablet-type food was produced in a usual manner such that one tablet was300 mg.

Example 15

[0077] (Drink Containing the Immunomodulator)

[0078] Purified water was added to two parts of enzymatically decomposedmaterial of Corynebacterum ammoniaphilum bacterial cells using egg-whitelysozyme and bromelain obtained in Example 8, 10 parts of cocoa butter,7 parts of granulated sugar, 7 parts of milk and 0.05 part ofemulsifying agent to adjust the total amount to 100 parts, and a cocoadrink was produced in a usual manner.

Example 16

[0079] (Treatment of Allergic Disorders)

[0080] Twenty-four patients, 18 to 47 years old, 16 males and 8 females,who suffered from pollinosis were divided into two groups consisting of12 persons. A hundred gram of cocoa drink, obtained in Example 13, wasdaily given for 6 months in one group. The other group was served as acontrol. Every persons of both groups was admitted wearing a cotton maskand occasional intake of medicines prescribed by the doctors of thepresent clinical study. Allergic status was evaluated at every 2 monthsfor each patient by the same doctor and scored symptoms about nasal,ocular, cutaneous, and respiratory signs. The scoring of the clinicalfeatures was normal (0), mild (1), moderate (2) and severe (3). TotalIgE level in the serum obtained at the start and the end of the studywere measured by RIST (radioimmunosorbent test) method.

[0081] The difference in the means of sum totals of clinical scoringbetween both groups was tested by one-tailed t-test. The differences ofIgE levels during 6 months before and after the treatment was comparedfor each group by two-tailed t-test. Significance was accepted atp<0.05. The results were summarized in Table 4.

[0082] The number of clinical scoring in the group ate theimmunomodulator was significantly lower than that of control group. IgElevels of this group was also significantly decreased. However, nosignificant decrease in the IgE level of the control group was observed.TABLE 4 Patient Sum of IgE (IU/ml) No. Treatment score Start End  1 Yes32 7070 5960  2 Yes 20  380  390  3 Yes 16 2309 2005  4 Yes 22  130  100 5 Yes 29 2248 2255  6 Yes 12  174  105  7 Yes 18  770  780  8 Yes 301110  526  9 Yes 20  145  30 10 Yes 13  640  650 11 Yes 19  120  25 12Yes 12  632  280 Mean ± SD 20.3 ± 6.7** 1310.7 ± 1968.3 1092.2 ± 1702.2*13 No 25  220  200 14 No 40 2100 1210 15 No 26  85  90 16 No 29  901 960 17 No 33  170 1900 18 No 38 1705 2185 19 No 38  88  204 20 No 20 260  130 21 No 28 8000 8226 22 No 22  68  130 23 No 32 5010 3480 24 No28  205  201 Mean ± SD 29.9 ± 6.2  1567.7 ± 2485.3 1576.3 ± 2352.8 

1. A bacterial cell wall decomposition product having animmunomodulative activity, which is produced by dissolving the cell wallof at least one selected from the group consisting of bacteria belongingto Corynebacteruim genera, Brevibacterium genera and Microbacteriumgenera and mutant strains of these bacteria.
 2. A process formanufacturing the bacterial cell wall decomposition product as definedin claim 1, which comprises dissolving the cell wall of at least oneselected from the group consisting of bacteria belonging toCorynebacteruim genera, Brevibacterium genera and Microbacterium generaand mutant strains of these bacteria.
 3. The process according to claim2, wherein the bacterial cell wall is digested with glucosidase and/orendopeptidase.
 4. The process according to claims 2 or 3, whichcomprises dissolving the cell walls with one of gulcosidase andendopeptidase in the medium of an isotonic solution, separating theprotoplast generated by the enzymatic digestion of the bacterial cellsfrom the medium, and furthermore enzymatically treating the dissolvedmaterial of the bacterial cells with the other of endopeptidase andglucosidase.
 5. The process according to any of claims 2 to 4, wherein abacterium of Corynebacteruim genus is Corynebacteruim glutamicum orCorynebacteruim herculis.
 6. The process according to any of claims 2 to4, wherein a bacterium of Brevibacterium genus is Brevibacterium flavum.7. The process according to any of claims 2 to 4, wherein a bacterium ofMicrobacterium genus is Microbacterium ammoniaphilum.
 8. The processaccording to claim 3 or 4, wherein the glycosidase is egg white lysozymeor N-acetylmuramidase SG.
 9. The process according to claims 2, whereinthe decomposition is performed by induction of lysogenic bacteriophage.10. The process according to claims 2, wherein the decomposition isperformed by autolysis.
 11. The process according to claim 3 or 4,wherein the endopeptidase is bromelain.
 12. The process according toclaim 3 or 4, wherein the endopeptidase is one selected from the groupconsisting of seratiopeptidase, protease produced by Streptomycesgriseus (K-1 strain), protease produced by Bacillus subtilis, Seaproze Sproduced by Armillaria mellea (Naratake in Japanese), trypsin,Achromobacter protease I and Grifola frondosa (Maitake in Japanese)metaloendopeptidase.
 13. The process according to claims 2 or 3, whichcomprises dissolving the cell walls with one of gulcosidase andendopeptidase in the medium of an isotonic solution, centrifuging thesolution to obtain the supernatant and enzymatically treating thesupernatant with the other of endopeptidase and glucosidase.
 14. AnImmunomodulator food or drink containing the bacterial cell walldecomposition product as defined in claim
 1. 15. An Immunomodulator foodor drink containing the bacterial cell wall decomposition product asobtained by the process as defined in claim
 2. 16. A method of treatingor preventing allergy, which comprises administering a pharmacologicallyeffective amount of the decomposition product as defined in claim 1 to aperson suffering from allergy.
 17. Use of the decomposition product asdefined in claim 1 for manufacturing immunomodulator.
 18. A method oftreating or preventing allergy, which comprises administering apharmacologically effective amount of at least one selected from thegroup consisting of bacteria belonging to Corynebacteruim genera,Brevibacterium genera and Microbacterium genera and mutant strains ofthese bacteria to a person suffering from allergy.
 19. Use of at leastone selected from the group consisting of bacteria belonging toCorynebacteruim genera, Brevibacterium genera and Microbacterium generaand mutant strains of these bacteria for manufacturing immunomodulator.20. An Immunomodulator food or drink containing at least one selectedfrom the group consisting of bacteria belonging to Corynebacteruimgenera, Brevibacterium genera and Microbacterium genera and mutantstrains of these bacteria.
 21. A method of effecting immunomodulation,which comprises administering a pharmacologically effective amount of atleast one selected from the group consisting of bacteria belonging toCorynebacteruim genera, Brevibacterium genera and Microbacterium generaand mutant strains of these bacteria to a person suffering fromimmunodeficiency.